Techniques for immobilizing bioactive proteins such as enzymes, antigens or antibodies onto an insoluble carrier such as a high molecular weight compound have been widely been studied as techniques for forming a bioreactor intended to separate, synthesize or decompose biosubstances. In recent years, however, utilization of the above-described immobilizing techniques for forming biosensors by immobilizing bioactive proteins onto the surface of a base of microelectrode or other transducer has been desired in addition to their utilization for forming bioreactors. For this purpose, it is generally required that the thin film functioning as carrier or support be thin and strong; that the immobilization be strong; that activity of the bioactive protein be kept at a high level; and that the immobilization amount be uniform. Further, in order to improve the response properties of the sensor, it is required to immobilize the bioactive protein onto a limited portion of a microtransducer at a high density.
In conventional enzyme-immobilizing processes, polymer beads or the like are generally used as carriers. However, few of them can form thin films showing good adhesion to a base due to their poor thin film-forming ability, because of their high molecular weights, or due to their swelling properties. In addition, since immobilization of enzymes is generally based on thermal reaction such as formation of amido bonds or Schiff bases, immobilization is difficult to control with respect to position or area.
JP-A-61-153559 (the term "JP-A" as used herein means an "unexamined published Japanese patent application") discloses a process of immobilizing a bioactive protein onto desired portions of a thin film formed on a sensor base by forming a pattern of a thin film containing the bioactive protein. In this process, a water-soluble diazide type light-sensitive polymer is used as a carrier, and a technique of hardening the polymer by irradiation with light to thereby immobilize enzyme dispersed in the polymer is employed.
This process is effective for the purpose of forming a pattern since the light-irradiating technique is employed for immobilization. However, since enzyme molecules are buried in the hardened resin, there is a considerable risk of losing enzyme activity. Further, highly active enzyme molecules located on the resin surface are considered to be easily desorbed.
JP-A-56-82093 discloses a process of dispersing an enzyme in an azido group-containing polymer and irradiating it with light to thereby immobilize the enzyme in the polymer matrix. In this process, however, water-soluble polymers (e.g., polyvinyl alcohol) are used to disperse the enzyme, and hence the light-crosslinked product has swelling properties, which makes it unsuited for forming a stable, insoluble film. In addition, because the enzyme molecules are buried in the polymer matrix as in the above described process, the enzyme activity is liable to be depressed.
It is important to solve these problems in order to form an organic thin film showing the high efficiency required for use as sensor, i.e., being strongly bound to a base and having a thin film thickness and a high immobilization density.